The thyroid gland concentrates inorganic iodine in blood plasma with the ultimate formation of two hormones 3,3',5'-triiodothyronine (T.sub.3) and 3,5,3',5'-tetraiodothyronine (thyroxine or T.sub.4) in an approximate 1:4 ratio. These hormones are transmitted through the circulatory system to cells where they regulate cell metabolism. In the circulatory system the hormones are in equilibrium with certain proteins in blood serum that bind up the hormone. These proteins are in the main globulin and, to a lesser extent prealbumin and albumin. For example, the equilibrium between T.sub.4 and thyroid hormone binding globulin (TBG) can be represented by the equation: EQU T.sub.4 +TBG.revreaction.T.sub.4 .multidot.TBG
a similar equilibrium for T.sub.3 also exists. Thus, in the blood at any given moment is a quantity of bound and unbound T.sub.3 and T.sub.4. The free hormone is thought to be the biologically active form.
In order to evaluate thyroid function, a number of tests have been developed which are designed to detect the unnatural conditions of hyperthyroidism and hypothyroidism. Among these tests are those designed to measure the total amount of the thyroid hormone T.sub.4 since normal limits have been established. An unnaturally high level of T.sub.4 is regarded as being indicative of hyperthyroidism whereas an unnaturally low amount indicates hypothyroidism.
Tests for total T.sub.4 are not completely reliable indicators of thyroid function. On the one hand, an amount of T.sub.4 higher than normal may not be clinically significant if the patient tested also has a higher than normal level of binding protein as occurs during pregnancy or when estrogen containing drugs are being used. On the other hand, a level of T.sub.4 within normal limits can exist when total binding protein is low because of liver disfunction and then fail to indicate hyperthyroidism.
In view of the shortcomings in tests for total T.sub.4, a complementary test that measures the binding capacity of the serum protein has been developed. This test is known as the T.sub.3 uptake test and is designed to measure the binding capacity of TBG in serum.
Both total T.sub.4 and T.sub.3 uptake tests are conveniently run using well known principles of radioassay. In a typical T.sub.3 uptake test, a known quantity of T.sub.3 that has been radioactively labeled, usually with I.sup.125 or I.sup.131, is admixed with a serum sample wherein it competes with naturally occuring thyroid hormone for binding sites. When equilibrium has been established, bound and unbound T.sub.3 are separated from the serum by adding an insoluble adsorbent for unbound T.sub.3 to the serum followed by separating the serum and adsorbent. Then the radiation emitted by either the serum containing the labeled and unlabeled TBG bound hormone or the adsorbent containing the unbound portion of the T.sub.3 is counted. The amount of radiation emitted is readily correlated to the binding capacity of TBG in the serum.
In a typical total T.sub.4 test procedure, the bound thyroxine in serum is separated from the binding proteins by denaturing the complex, for example with alcohol which precipitates the binding proteins leaving about 80% of the T.sub.4 in solution in the serum. The serum containing thyroxine is mixed with a buffered solution of TBG and a known amount of labeled T.sub.4. The serum T.sub.4 and labeled T.sub.4 compete for the limited amount of TBG. The sample is then contacted with a suitable insoluble adsorbent for non-TBG bound labeled and unlabeled T.sub.4. The serum containing TBG bound T.sub.4 and adsorbent are separated and the radiation emitted by one or the other is counted. The amount of radiation emitted is a function of the total T.sub.4 in the serum sample.
In both the T.sub.3 uptake test and test for total T.sub.4, the adsorbent plays a vital role as both tests demand efficient separation between hormone that is bound to protein and free hormone if reproducible accuracy is to be achieved. Thus, among the commercially available diagnostic kits, the principal difference lies in the choice of adsorbent. Adsorbents that have been used include ion exchange resins, for example those disclosed in U.S. Pat. No. 3,414,383. As adsorbents, ion exchange resins suffer from the disability that their avidity for thyroid hormone is so high that they begin to strip thyroid hormone from the TBG or other protein. In addition, a long time, i.e., up to one hour, is required to reach equilibrium. Ion exchange resins also lack a clear end point to distinguish when free hormone is removed and when bound hormone is being stripped from the TBG. There is also an effect of serum aging on the performance of resin tests due to the release of organic acids which can compete with thyroid hormones for binding sites on the resin. See Shaw, W. Hubert, I. L., and Spierto, F. W., Clin. Chem., 22, 673 (1976).
Sephadex, a non-ionic resin gel of crosslinked dextran has also been used. See Murphy and Pattee, J. Clin. Endo., 24, 187 (1964). It has a low affinity for unbound thyroid hormone and, like the ionic exchange resins, requires long equilibrium times.
Other suggested adsorbents are the particulate inorganic crystalline materials described in U.S. Pat. No. 3,666,854. The preferred member of this group is the magnesium silicate known as talc. Equilibrium is rapidly reached using this adsorbent but process variables, i.e., technique of mixing and shaking to keep the serum and talc in contact can result in variations. A principal problem is keeping the talc suspended in the serum as it settles out quickly.
Another proposed method is based upon charcoal as an adsorbent. The charcoal is first treated with a large molecule such as hemoglobin, dextran or the like to fill in the large pores that would adsorb TBG bound hormone as well as the unbound. Charcoal suffers from the disadvantage that equilibrium is never reached and it strips hormone from the binding protein. References to charcoal based techniques are summarized in U.S. Pat. No. 3,721,528. No commercially available test kit presently uses a charcoal adsorbent to measure T.sub.3 uptake or total T.sub.4 by competitive protein binding.
Another material proposed for use as an adsorbent is microspherical albumin. See Rolleri, et al J. Nucl. Med., 13, 893 (1972). Albumin microspheres are difficult to make, their preparation literally requires the "boiling in oil" of albumin, and are thus economically unattractive. The spheres also have a very low avidity for thyroid hormone requiring a prolonged equilibration time and are difficult to keep in suspension. Thus, for several reasons the microspheres of albumin are not well suited as the adsorbent in a diagnostic test kit.
Many of the adsorbents used in diagnostic kits for evaluating thyroid function are also used in radioassay based on similar techniques where it is necessary to adsorb a component from a body fluid. Thus, the shortcomings they display in that use also occur in other radioassay tests.
From the foregoing it can be seen that the processes known to the prior art for separating adsorbable components from a body fluid are limited in their effectiveness by the shortcomings of the adsorbents previously employed.